BINDING OF MONOCLONAL ANTI-NATIVE DNA AUTOANTIBODIES TO DNA OF VARYING SIZE AND CONFORMATION
BINDING OF MONOCLONAL ANTI-NATIVE DNA AUTOANTIBODIES TO DNA OF VARYING SIZE AND CONFORMATION
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DOI：
10.1016/0161-5890(85)90065-3
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发表时间：
1985-01-01
影响因子：
3.6
通讯作者：
STOLLAR, BD
中科院分区：
文献类型：
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作者：
ALI, R;DERSIMONIAN, H;STOLLAR, BD
磷的微量化学测定被应用于测量由单克隆或血清抗DNA自身抗体和不同大小和构象的DNA形成的免疫复合物中的DNA。两种单克隆抗体由源自自身免疫MRL-lpr/lpr小鼠脾细胞的杂交瘤产生，并通过在山羊抗小鼠Ig-Sepharose柱上的亲和层析从培养液中纯化。通过用微球菌和 S1 核酸酶短暂消化小牛胸腺 DNA 并在 Sepharose 4B 上分级分离来制备双螺旋 DNA 片段；通过热变性测量证实了它们的双链结构。用单克隆或血清抗体与天然DNA或DNA片段或变性DNA形成免疫复合物；用山羊抗小鼠IgG沉淀复合物并洗涤，测定沉淀物的DNA磷含量。对于一种单克隆自身抗体 (H241)，随着 DNA 大小的增加，可结合的 DNA 量会不连续增加（并且半最大结合所需的抗原浓度会减少）。随着片段大小从平均 100 个（范围 85-105）碱基对增加到平均 150 个（范围 105-170）碱基对，并再次增加到 450 个（范围 360-620）和 600 个（范围 425-825）碱基对，结合效率尤其显着增加。第二种单克隆抗体 (H143) 在与大于 300 个碱基对的 DNA 片段的结合方面没有表现出显着变化。对于较小的片段，H143 结合的 DNA 量减少，但半最大结合所需的 DNA 浓度却没有减少。这些单克隆抗体的亲和力在人系统性红斑狼疮血清 IgG 抗 DNA 自身抗体的范围内。结合力对摩尔的依赖性。 wt 对于评估这些单克隆抗体作为生化试剂以及作为体内免疫复合物形成的潜在参与者非常重要。
A microchemical assay for phosphorus was applied to the measurement of DNA in immune complexes formed with monoclonal or serum anti-DNA autoantibodies and DNA of varying size and conformation. Two monoclonal antibodies were produced by hybridomas derived from spleen cells of autoimmune MRL-lpr/lpr mice and were purified from culture fluid by affinity chromatography on columns of goat anti-mouse Ig-Sepharose. Double-helical DNA fragments were prepared by brief digestion of calf thymus DNA with micrococcal and S1 nucleases and fractionation on Sepharose 4B; their double-strand structure was confirmed by measurement of thermal denaturation. Immune complexes were formed with monoclonal or serum antibodies and native DNA or DNA fragments or denatured DNA; the complexes were precipitated with goat anti-mouse IgG and washed, and DNA phosphorus content of the precipitates was measured. With one monoclonal autoantibody (H241), there were discontinuous increases in the amount of DNA that could be bound (and decreases in the antigen concn required for half-maximal binding) as the DNA size increased. There were especially marked increases in binding efficiency as fragment size increased from an average of 100 (range 85-105) to an average of 150 (ranging 105-170) base pairs, and again between 450 (range 360-620) and 600 (range 425-825) base pairs. A second monoclonal antibody (H143) did not show significant variation in binding with DNA fragments larger than 300 base pairs. With smaller fragments, the amount of DNA bound by H143 was reduced, but the DNA concn required for half-maximal binding was not. Affinities of these monoclonal antibodies were within the spectrum of human systemic lupus erythematosus serum IgG anti-DNA autoantibodies. The dependence of binding on mol. wt is important in the evaluation of these monoclonal antibodies as biochemical reagents and as potential participants in formation of immune complexes in vivo.